Retroviruses are RNA viruses that replicate through a DNA intermediate. This large family of viruses is found to infect all vertebrates and includes gammaretroviruses, lentiviruses and spumaviruses. Currently, replication-deficient vectors derived from gammaretroviruses or lentiviruses represent the most frequently used tool for stable viral gene transfer. These vectors have a reasonable genetic payload (up to 9 kb), high transducing efficiencies both in vitro and in vivo, and the ability to permanently modify the genetic content of the target cell. Classically, retroviral vector production involves either transient transfection of multiple DNA plasmids encoding required components for viral packaging or alternatively the use of a packaging cell line which stably expresses the proteins required for viral assembly. In both cases, the manipulated cells produce viral particles that are subsequently recovered in the cell supernatant. Although both of these approaches permit production of functional viral vectors, the titers obtained are relatively low, making it difficult to scale production and efficiently obtain large titers. In addition, current processes are often laborious, time consuming and lack robustness. Thus, there is a need for a method to obtain cells containing stably integrated retroviral vectors without the need to pass by a discrete viral vector production process.
Spumaviruses or Foamy viruses (FV) are a subfamily of retroviruses that are endemic to most non-human primates, horses, cattle and cats (Saib 2003; Switzer, Salemi et al. 2005). The foamy virus (FV) replication pathway has been shown to differ from the classical retroviral pathway. FV infection starts with attachment to target cells and binding to an, as yet unknown, but potentially very ubiquitous cellular receptor. Upon arrival of capsids into the cytoplasm, they seem to dock to dynein motor protein complexes and migrate along microtubules towards the microtubule organizing center (MTOC) where they accumulate. Further, the disassembly apparently involving capsid processing by viral and cellular proteases occurs. Before the viral integration, the FV pre-integration complex is localized in the nucleus. Expression of FV genes by the cellular transcription machinery is regulated through a viral transactivator utilizing internal and LTR derived promoter elements. Then, spliced RNAs are exported out of nucleus and FV accessory, capsid and enzymatic genes are translated in the cytoplasm, whereas envelope glycoproteins are translated at the rough ensoplasmatic reticulum to target the secretory pathway. The FV assembly involves transport of Gag to the MTOC where a preassembly of capsids takes place. Unlike orthoretrovirus (a subfamily including gammaretroviruses and lentiviruses), FVs reverse transcribe their encapsidated RNA genome during assembly and/or budding, leading to the production of DNA containing virions. The ability to generate cDNA before budding of the virion has been shown to allow recycling of the genome into the nucleus resulting in intracellular retrotransposition (Heinkelein, Pietschmann et al. 2000; Pietschmann, Zentgraf et al. 2000). In addition, viral particles can also be released into the environment, or transferred by a cell-to-cell mechanism (Heinkelein, Pietschmann et al. 2000; Pietschmann, Zentgraf et al. 2000) (See FIG. 1).
Although foamy viruses were only more recently introduced into the repertoire of vector systems for the correction of inherited diseases (in particular the hematopoietic lineage in mammals) they represent an attractive alternative to gammaretroviruses and lentiviruses, displaying several additional advantages including the absence of FV antibodies in the human population, the benign course of natural FV infections, their very broad host cell range, a safer (i.e. more random) integration profile and an extended packaging limit (12 kb) (Lindemann and Rethwilm 2011).